1. Field of the Invention
This invention relates to the design of a double antigenically marked classical swine fever virus (CSFV) live attenuated candidate strain vaccine validated for absence of reversions. The FlagT4-mFT-Gv virus is a modified recombinant FlagT4 RB-C22 virus containing a multiple-mutated Flag insertion epitope in the E1 glycoprotein and a multiple-mutated WH303 epitope in the E2 glycoprotein.
2. Description of the Relevant Art
Classical swine fever (CSF) is a highly contagious disease of swine. The etiological agent, CSF virus (CSFV), is a small, enveloped virus with a positive, single-stranded RNA genome, classified as a member of the genus Pestivirus within the family Flaviridae (Becher et al. 2003. Virology 311: 96-104). The 12.5 kb CSFV genome contains a single open reading frame that encodes a 3898-amino-acid polyprotein and ultimately yields 11 to 12 final cleavage products (NH2-Npro-C-Ems-E1-E2-p7-N52-N53-NS4A-NS4B-NS5A-NS5B-COOH) through co- and post-translational processing of the polyprotein by cellular and viral proteases (Rice, C. M. 1996. In: Fundamental Virology, 3rd edition, Fields and Howley, eds., Lippincott Raven, Philadelphia, pp. 931-959). Structural components of the CSFV virion include the capsid (C) protein and glycoproteins Ems, E1, and E2. The E1 and E2 glycoproteins are anchored to the envelope at their carboxyl termini and Ems loosely associates with the viral envelope (Thiel et al. 1991. J. Virol. 65: 4705-4712; Weiland et al. 1990. J. Virol. 64: 3563-3569; Weiland et al. 1999. J. Gen. Virol. 80: 1157-1165). E1 and E2 are type I transmembrane proteins with an N-terminal ectodomain and a C-terminal hydrophobic anchor (Thiel et al., supra). E2 is the most immunogenic of the CSFV glycoproteins (Konig et al. 1995. J. Virol. 69: 6479-6486; van Gennip et al. 2000. Vaccine 19:447-459); Weiland et al. 1990, supra), inducing neutralizing antibodies, which provide protection against lethal CSFV challenge.
The two main policies used for CSFV control are prophylactic vaccination or non-vaccination with “stamping out” of exposed animals in the event of an outbreak. Countries considered free of CSF do not recommend the use of currently available live attenuated viruses as tools to control outbreaks of the disease, despite the proven efficacy of these vaccines in eliciting a rapid and solid protection against the virus (van Oirschot, J. T. 2003. Vet. Microbiol. 96: 367-384). The humoral immune response induced by these vaccines does not differ from that elicited by infections caused by wild-type viruses; hence, the use of CSFV live attenuated viruses has been hampered by their inability of inducing a response differentiable between infected and vaccinated animals, i.e., by their lack of DIVA capability. Thus, the use of a CSFV live attenuated virus with DIVA capabilities could significantly impact policies of disease control. CSFV subunit marker vaccines with DIVA capabilities have been developed using recombinant CSFV E2 envelope protein (Hulst et al. 1993. J. Virol. 67: 5435-5442; Van Rijn et al. 1996. J. Gen. Virol. 77: 2737-2745; Van Rijn et al. 1999. Vaccine 17: 433-440)). The onset of immunity elicited by subunit vaccines occurs two weeks post-vaccination, limiting their efficacy relative to traditional live attenuated virus vaccines when animals are exposed to CSFV shortly after vaccination (Bouma et al. 2000. Vaccine 18: 1374-1381; Uttenthal et al. 2001. Vet. Microbiol. 83: 85-106).
Recently, we reported the development of a CSFV experimental marker live attenuated virus strain, FlagT4v (Holinka et al. 2009. Virology 384:106-113). FlagT4v contains the synthetic epitope of Flag® (Sigma, St. Louis, Mo.) as an insertion within the 19 mer insertion of a previously modified E1. Flag® serves as a positive antigenic marker. FlagT4 also serves as a negative antigenic marker as the result of the abolition of a highly conserved CSFV-specific epitope recognized by monoclonal antibody WH303 (mAbWH303e) (Edwards et al. 1991. Vet Microbiol. 29:101-108). Immunization with FlagT4v induced complete protection against challenge with virulent CSFV Brescia, both at 3 and 28 days post-infection (DPI). Serological responses against both the Flag and mAbWH303 epitopes in animals immunized with FlagT4v allowed the discrimination of animals immunized with FlagT4v from animals challenged with CSFV Brescia.
FlagT4 was further analyzed as a candidate vaccine strain and its minimal protective dose, biosafety and attenuation stability were determined. Although FlagT4v was completely atoxic and showed a protective efficacy compatible with further development as a vaccine, it presented some degree of reversion to virulence when successively passed in swine. Thus, there was a need to modify the FlagT4 virus to obtain an attenuated virus that has the attributes of a positive and a negative marker and is also a successful live attenuated vaccine.